Sun-Young Ju

Mesenchymal stem cells showed the highest potential for the regeneration of injured liver tissue compared with other subpopulations of the bone marrow

We have previously reported that bone marrow cells (BMCs) participate in the regeneration after liver injury. However, it is not established that this is the result of differentiation of hematopoietic stem cells (HSCs), mesenchymal stem cells (MSCs) or the combination of both. We investigated the contribution of each cell fraction to the regenerative process. First, we confirmed that transplanted stem cells migrate directly to injured liver tissue without dispersing to other organs. Next, we divided green fluorescent protein (GFP)‐expressing BMCs into three populations as mononuclear cells, MSCs and HSCs. We then compared the engraftment capacity after transplantation of each fraction of cells into liver‐injured mice. Of these, the MSCs transplanted group showed the highest GFP fluorescence intensities in liver tissue by flow cytometry analysis and confocal microscopic observation. Furthermore, MSCs showed differentiation potential into hepatocytes when co‐cultured with injured liver cells, which suggests that MSCs showed highest potential for the regeneration of injured liver tissue compared with those of the other two cell refractions.

MSC–DC interactions: MSC inhibit maturation and migration of BM-derived DC

BACKGROUND Mesenchymal stromal cells (MSC) comprise one of the BM stromal cells that are known to support hematopoiesis. It has also been suggested recently that MSC display immunosuppressive capacities through inhibiting the differentiation of monocyte-derived DC. DC travel to the lymph nodes (LN) to present Ag to T cells, and CCL21 is the chemokine that plays an important role in DC migration into the T-cell area of LN. We addressed the effect of MSC on this chemotactic activity of DC, one of the typical characteristics upon maturation. METHODS BM cells were isolated and then cultured for generation of myeloid DC in the presence of GM-CSF and/or lipopolysaccharide with or without MSC. MSC were identified by flow cytometry of the immunologic markers and by performing colony-forming unit fibroblast assay. Migration of DC was observed with a newly developed time-lapse video microscopic technique. RESULTS MSC co-culture inhibited the initial differentiation of DC, as well as their maturation. The matured DC actively migrated directionally in response to CCL21, a powerful DC-attracting chemokine, whereas the MSC co-cultured DC did not. DISCUSSION Collectively, the findings of these experiments raise the possibility that MSC suppress the migratory function of DC and so they may serve immunoregulatory activities through the modulation of the Ag-presenting function of DC.

Effect of hypoxic treatment on bone marrow cells that are able to migrate to the injured liver

Restricted numbers and poor regenerative properties limit the use of adult stem cells. We tested the effect of hypoxic treatment as a method by which to increase cell migration. Bone marrow cells (BMCs) were cultured under oxygen saturations of 0.1, 3, and 20% for 24 h. After hypoxic treatment, BMCs of apoptotic fraction were decreased. The expression of CXCR4 was noticeably increased in the hypoxia‐treated BMCs and their migration in response to SDF‐1α was enhanced compared with cells cultured under normoxic condition. Hypoxic BMCs had a higher degree of engraftment to the CCl4‐injured liver than the normoxic cells. Hypoxic treatment of BMCs may have merits in decreasing apoptosis of those cells as well as in enhancing cellular migration to SDF‐1α, the chemokine which binds to BMCs expressed CXCR4 and to the injured tissue, such as CCl4 damaged liver.

Effect on Cell Cycle Progression by N-Myc Knockdown in SK-N-BE(2) Neuroblastoma Cell Line and Cytotoxicity with STI-571 Compound

PURPOSE Neuroblastoma is a common tumor in childhood, and generally exhibits heterogeneity and a malignant progression. MYCN expression and amplification profiles frequently correlate with therapeutic prognosis. Although it has been reported that MYCN silencing causes differentiation and apoptosis in human neuroblastoma cells, MYCN expression influences the cytotoxic potential of chemotherapeutic drugs via the deregulation of the cell cycle. STI-571 may constitute a promising therapeutic agent against neuroblastoma, particularly in cases in which c-Kit is expressed preferentially in MYCN-amplified neuroblastoma. MATERIALS AND METHODS To determine whether STI-571 exerts a synergistic effect on cytotoxicity with MYCN expression, we assessed apoptotic cell death and cell cycle distribution after 72 h of exposure to STI-571 with or with out treatment of SK-N-BE(2) neuroblastoma cells with MYCN siRNA. RESULTS MYCN siRNA-treated SK-N-BE(2) cells did not affect apoptosis and cells were arrested in G0/G1 phase after STI-571 treatment. CONCLUSIONS siRNA therapy targeted to MYCN may not be effective when administered in combination with STI-571 treatment in cases of neuroblastoma. Therefore, chemotherapeutic drugs that target S or G2-M phase may prove ineffective when applied to cells arrested in the G0/1 phase as the result of MYCN knockdown and STI-571 treatment.

Mesenchymal stem cells inhibit the differentiation of CD4+ T cells into interleukin-17-secreting T cells.

and might possibly be one of the immunoregulatory mechanisms exploited by MSCs. Sixto eight-week-old C57BL/6 female mice were killed by cervical dislocation and limbs were removed. The bone marrow was flushed from the medullary cavities of both the femurs and tibias with serum-free RPMI 1640 (Gibco BRL, Carlsbad, Calif., USA) medium using a 25-gauge needle, filtrated through Nylon meshes and centrifuged for 5 min at 1,200 rpm. Isolated bone marrow cells were then incubated in RBC lysis solution (0.15 M NH 4 Cl, 10 m M NaHCO 3, 10 m M EDTA) and washed twice with phosphate-buffered saline. The cells were then plated at 1 ! 10 7 cells/100-mm culture dishes in Iscove’s modified Dulbecco’s medium (Sigma, St. Louis, Mo., USA) with 10% heat-inactivated fetal bovine serum. After 48 h, nonadherent cells were removed via aspiration and the medium, consisting of MesenCult TM basal medium and 10% MSC stimulatory supplement (Stemcell Technologies, Vancouver, Canada), was replenished. We cultured plastic adherent cells for more than 3 weeks in MSC medium. After 14 days, colony-forming units of fibroblasts were observed and confirmed after Giemsa staining ( fig. 1 ). For adipogenic differentiation, MSCs were cultured for 3 weeks in MesenCult basal medium containing 10% adipogenic stimulatory supplement (Stemcell Technologies). Total CD4+ cells were prepared from the lymph nodes and spleen (purity 1 98%, 3 ! 10 6 cells/mouse) using magnetic beads (clone L3T4, Miltenyi Mesenchymal stem cells (MSCs) are a subset of stem cells which reside principally in bone marrow and can be isolated from a wide variety of adult and fetal tissues. MSCs can be differentiated into multiple mesodermal tissues, including osteoblasts, chondrocytes and adipocytes, providing promising cellular resources for the repair of tissues. In addition to their differentiation potential, MSCs have been reported to exhibit immunoregulatory functions via the suppression of T cells, B cells, NK cells and dendritic cell functions [1, 2] . As MSCs evidence such unique immunoregulatory properties, it may be possible to employ MSCs as therapeutic tools for immune-mediated diseases. The immunomodulatory effect of MSCs on T cells is involved in the inhibition of proliferation and cytokine production, as well as the enhancement of MSC immunosuppressive activity via T cell-derived cytokines. T helper 17 (Th17) cells were identified as a new arm of an effector T cell lineage which can be induced by transforming growth factor (TGF)and interleukin (IL)-6 stimulation and can generate IL-17 [3] . TGFand IL-6 induce Th17 cells, and IL-23 may be crucial for the maintenance of Th17 pools in chronic inflammation. Although IL-17 may be involved in the recruitment of neutrophils, eradicating extracellular bacteria and chronic inflammation, it has also been reported that IL-17A enhanced the proliferation of MSCs [4] . From these fundamental facts, we hypothesized that MSCs might exert an effect on the differentiation of Th17 cells Received: September 1, 2008 Accepted after revision: October 2, 2008 Published online: November 28, 2008

Gene expression profile of mesenchymal stromal cells after co-culturing with injured liver tissue

Mesenchymal stromal cells (MSCs) are a potential cell source for the development of therapeutic products. Recent studies have shown that the transplantation of MSCs to damaged organs, including the heart, liver and kidneys, results in the restoration of the damaged tissues. However, the mechanisms underlying this regeneration process have yet to be clearly characterized. Consequently, in this study, we focused on the therapeutic potential of MSCs in injured liver tissue by evaluating the gene expression profiles of MSCs in the presence of injured liver and normal liver cells using a microarray chip containing 44,000 genes. In order to mimic the state of liver cell regeneration in vitro, we respectively co-cultured MSCs with CCl4-injured liver cells and normal liver cells from C57BL/6 female mice. After 48 h of co-culturing, MSCs were collected and the RNA was extracted for microarray analysis. Under conditions of co-culture with normal liver cells, upregulated expression of CXCR6, CCR3, IL-2, IL-11, CD34, CD74, procollagen, FMS-like tyrosine kinase, neuregulin 4, Wnt2 and catenins was noted. Under conditions of co-culture with the CCl4-injured liver cells, expression of CXCL2, cytoglobin, erythropoietin, v-Erb, hypoxia-inducible factor 3 (α subunit), retinoic acid receptor β and Vav2 was upregulated. Our research provides information regarding the differential molecular mechanisms that regulate the properties of MSCs in the regeneration of injured liver tissue.

Hypoxia Affected SDF-1α-CXCR4 Interaction between Bone Marrow Stem Cells and Osteoblasts via Osteoclast Modulation

enzymatic cleavage of the linkage between OBs and HSCs. OCLs activated by stress-induced signals secrete proteolytic enzymes such as matrix metalloproteinase 9 (MMP9) and cathepsin K (CTK) that are responsible for the degradation of bone mineral and collagen matrix. These enzymes trigger weakness in the OBs-HSCs anchorage and ultimately lead to the mobilization of stem cells into circulation. Among the molecules which might be weakened by OCLs in the osteoblastic niche, we focused on SDF-1 , a molecule expressed on OBs and its cognitive receptor, CXCR4, expressed on the HSCs [5] . SDF-1 , the most well-known powerful stem cell chemoattractant, is a stem cell survival factor and also a regulator of interactions facilitating the adhesion between stem cells and the extracellular matrix or stromal cells [6] . These observations indicate that the SDF-1 -CXCR4 axis may be involved in regulating the HSCs residence in niche. Hypoxia is a state of reduced or inadequate oxygen supply, and common feature of inflammation, injury, and tumors [7] and these hypoxic responses are maintained via hypoxia-inducible factor-1 (HIF-1 ) stabilization, a subunit of HIF-1 and an oxygen-sensitive transcription factor, as well as HIF-2 and NFB [8, 9] . Low oxygen tension physiologically influences various cell types including OCLs [10] and this affects stem cell niche in the BM [11] . We attempted to determine whether hypoxia can affect the regulation of the stem cell niche by enhancing OCL activation in the BM. For OCLs, RAW 264.7 mouse monocyte/macrophage cells were purchased from American Type Culture ColOsteoclasts (OCLs) are bone-resorbing multinucleated cells derived from macrophage-monocyte lineage progenitors. These tissue-specific, specialized cells require receptor activator of nuclear factorB ligand (RANKL) signals from osteoblasts (OBs) for their proliferation, differentiation and bone-resorbing activity. Responding to RANKL stimulation, bone marrow (BM) OCL precursors become functional OCLs and play a central role in the regulation of bone mass along with bone-forming OBs [1] . Recently, it was reported that OCLs can lead to stem cell mobilization as a novel component of a stem cell niche in the BM. In mammals, BM composed mainly of hematopoietic stem cells (HSCs) is encased within the bone structure. A portion of these hematopoietic cells, classified as an osteoblastic niche, can be found next to the endosteal bone surface, a surface lined primarily by OBs [2] . This anatomical arrangement makes it possible for reciprocal communication between the two cell types. To maintain physiological homeostasis, the niche orchestrates a myriad of signaling and adhesive interactions through the linkage between stem cell factor/c-Kit, Jagged/Notch, angiopoietin-1/Tie2, and Ca 2+ -sensing receptor [3] . All of the cell surface receptors for these signals have been found on HSCs, and their respective ligands are mostly expressed by osteoblastic cells of the endosteal bone niche. Ligation of these receptors and ligands maintains the adhesive interaction of the steady state; however, under stress situations such as inflammation, injury and hypoxia, these balances are broken and HSCs are mobilized into the bloodstream [4] . During this process, OCLs play a role in the Received: August 20, 2009 Accepted after revision: September 30, 2009 Published online: December 2, 2009